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|Title:||The N-degron approach to create temperature-sensitive mutants in Schizosaccharomyces pombe|
|Citation:||Rajagopalan, S., Liling, Z., Liu, J., Balasubramanian, M. (2004-07). The N-degron approach to create temperature-sensitive mutants in Schizosaccharomyces pombe. Methods 33 (3) : 206-212. ScholarBank@NUS Repository. https://doi.org/10.1016/j.ymeth.2003.11.015|
|Abstract:||Conditional mutants are a vital tool for analysis of gene function. The use of temperature-sensitive mutants in Schizosaccharomyces pombe has significantly promoted understanding of many cellular processes. A portable heat-inducible amino-terminal degron (N-degron) for conditional degradation of a gene product has been previously described in Saccharomyces cerevisiae. This paper describes the adaptation of the N-degron method to create temperature-sensitive (ts) mutants in S. pombe. A ts derivative of the mouse dihydrofolate reductase with an amino-terminal arginine (Arg-DHFRts) previously described in S. cerevisiae was fused to the N-terminus of Bir1p, a nuclear protein involved in mitotic chromosome segregation in S. pombe. This fusion allele, referred to as bir1-td, conferred a chromosome segregation defect at 36°C, as with previously described alleles of bir1. Deletion of the S. pombe E3 ubiquitin ligase (N-recognin), Ubr11p, reversed the temperature-dependent lethality of bir1-td, providing evidence for N-end rule mediated destruction of Bir1p. The methods we describe should therefore facilitate analysis of essential genes in fission yeast for which conditionally lethal mutants are unavailable. © 2003 Elsevier Inc. All rights reserved.|
|Appears in Collections:||Staff Publications|
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